Two approaches were used to isolate fragments of chitin synthase genes from the opportunistic human pathogen Aspergillus fumigatus. Firstly, regions of amino acid conservation in chitin synthases of Saccharomyces cerevisiae were used to design degenerate primers for amplification of portions of related genes, and secondly, a segment of the S. cerevisiae CSD2 gene was used to screen an A. fumigatus lambda genomic DNA library. the polymerase chain reaction (PCR)-based approach led to the identification of five different genes, designated chsA, chsB, chsC, chsD and chsE. chsA, chsB, and chsC fall into Classes I, II and III of the 'zymogen type' chitin synthases, respectively. The chsD fragment has approximately 35% amino acid sequence identity to both the zymogen type genes and the non-zymogen type CSD2 gene. chsF appears to be a homologue of CSD2, being 80% identical to CSD2 over 100 amino acids. An unexpected finding was the isolation by heterologous hybridization of another gene (chsE), which also has strong sequence similarity (54% identity at the amino acid level over the same region as chsF) to CSD2. Reverse transcriptase-PCR was used to show that each gene is expressed during hyphal growth in submerged cultures.